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Plant Physiol. (1 996) 1 12: 1479-1 490

Red Xylem and Higher Lignin Extractability by Down- Regulating a Cinnamyl Alcohol Dehydrogenase in Poplar’

Marie Baucher, Brigitte Chabbert, Cilles Pilate, Jan Van Doorsselaere, Marie-ThCrPse Tollier, Michel Petit- Conil, Daniel Cornu, Bernard Monties, Marc Van Montagu*, Dirk InzC, Lise Jouanin, and Wout Boerjan

Laboratorium voor Genetica, Department of Genetics, Flanders lnteruniversity lnstitute for Biotechnology (M.B., J.V.D., M.V.M., D.I., W.B.), and Laboratoire Associe de I’lnstitut National de Ia Recherche Agronomique (France), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000, Belgium (M.B., D.I.); Laboratoire de Chimie

Biologique, lnstitut National de Ia Recherche Agronomique, F-78850 Thiverval-Grignon, France (B.C., M.-T.T., B.M.); Station d’Amélioration des Arbres Forestiers, lnstitut National de Ia Recherche Agronomique, F-45 1 60 Ardon, France (G.P., D.C.); Centre Technique du Papier, Domaine Universitaire, B.P. 251, F-38044 Grenoble

Cedex 09, France (M.P.-C.); and Laboratoire de Biologie Cellulaire, lnstitut National de Ia Recherche Agronomique, F-78026 Versailles Cedex, France (L.J.)

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step i n the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Popuhs tremula x Populus alba) by both antisense and co-suppression strategies. Sev- era1 antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCI staining was different i n the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. More- over, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were detected in the soluble alkali fraction of the CAD down-regulated poplars. Alkaline pulping experiments on 3-month-old trees showed a reduction of the kappa number without affecting the degree of cellulose degradation. These results indicate that reducing the CAD activity i n trees might be a valuable strategy to optimize certain processes of the wood industry, especially those of the pulp and paper industry.

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Lignin is an integral cell wall component of a11 vascular plants (Grisebach, 1981). It plays an important role in plant cell walls, enhancing the rigidity, conferring resistance toward pathogens and mechanical stress, and enabling solute transport in the xylem (Lewis and Yamamoto, 1990).

This work was supported by grants from the Commission of the European Communities (ECLAIR-OPLIGE no. AGRE-0021-C [EDB] and FAIR-CT95-0424). M.B. and J.V.D. are indebted to the European Molecular Biology Organization and the European Union for short-term fellowships and to the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onder- zoek in de Industrie for a postdoctoral fellowship, respectively. D.1. is a research director of the Institut National de la Recherche Agronomique (France).

* Corresponding author; e-mail mamon8genwetl .rug.ac.be; fax 32-9 -2645349.

Among woody plants, the amount of lignin varies between 15 and 36% of the dry weight (Higuchi, 1985). Despite its biological importance in plants, lignin is an undesirable component in the pulp and paper industry because it must be removed from the wood fibers (Whetten and Sederoff, 1991; Dean and Eriksson, 1992); this process consumes large quantities of energy and hazardous chemicals. Re- ducing the amount or changing the quality of lignin in trees would be beneficia1 from an economical as well as an environmental point of view. Also, lignin decreases forage crop digestibility (Akin and Chesson, 1989).

Lignin is a complex macromolecule that originates from the oxidative polymerization of cinnamyl alcohols as prin- cipal monomeric units (Fig. 1). These lignin monomers (or monolignols) are the p-coumaryl, coniferyl, and sinapyl alcohols. Lignin molecules are generally classified into three major groups based on their structural units. Woody angiosperm lignin consists of both coniferyl and sinapyl alcohols and is classified as guaiacyl-syringyl lignin, whereas gymnosperm lignin contains mainly coniferyl alcohol and is classified as guaiacyl lignin. Grasses incorpo- rate the three types of monomers in their lignin, which is called p-hydroxyphenyl-guaiacyl-syringyl lignin (Higuchi, 1985). Furthermore, variations in lignin structure and monomeric composition, depending on plant species, cytolog- ical origin, conditions of growth, and stage of development, have been recognized and described as lignin heterogeneity (Monties and Lapierre, 1981; Fengel and We- gener, 1984; Aloni et al., 1990; Sakakibara, 1991; Campbell and Sederoff, 1996).

Representatives of most of the genes of the lignin biosynthesis pathway have been cloned and characterized (for reviews, see Boudet et al., 1995; Whetten and Sederoff, 1995; Boerjan et al., 1996), which has allowed researchers to modify the lignin quality and quantity in plants. Trans-

Abbreviations: AF, alkali fraction; AL, alkali lignin; CAD, cinnamyl alcohol dehydrogenase; COMT, caffeic acid/5- hydroxyferulic acid O-methyltransferase; CWR, cell wall residue; DP, degree of polymerization; SR, saponified residue.

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1480 Baucher et al. Plant Physiol. Vol. 112, 1996

O w O H O V O H

PA L NH,

c- henylalanine

O w O H O y O H 0-OH O v O H O V O H

OH p-coumaric caffeic ferulic 5-hydroxyferulic sinapic

acid acid acid acid acid

1 4 C L 1 4 C L 1 4 C L

1 CCR 1 CCR 1 CCR H

B

CH, OH O H

p-coumaraldehyde coniferaldehyde sinapaldehyde

1 CAD 1 CAD 1 CAD

OH OCH,

p-coumaryl coniferyl sinapyl alcohol alcohol alcohol

(H) IGI ISI

1 / Peroxidases / Laccases

LlGNlNS

Figure 1. Lignin biosynthesis pathway. PAL, Phe ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H, coumarate 3-hydroxylase; F5H, ferulate 5-hydroxylase; 4CL, 4-coumarate-COA ligase; and CCR, cinnamoyl-COA reductase.

\

\ I /

genic tobacco plants with a 75% decreased Phe ammonia- lyase activity had a reduced lignin content. However, these plants, in addition to having lower levels of phenylpropanoid products, were also severely affected in their development (Elkind et al., 1990; Bate et al., 1994). Severa1 groups have down-regulated bispecific COMT in tobacco (Dwivedi et al., 1994; Ni et al., 1994; Atanassova et al., 1995) and in poplar (Populus; Van Doorsselaere et al., 1995a). In the transgenic plants with the most reduced COMT activity, a drastic decrease in the syringyl/guaiacyl ratio and the appearance of the 5-hydroxyguaiacyl unit in the lignin were observed, but there was no alteration of the lignin content (Atanassova et al., 1995; Van Doorsselaere et al., 1995a). An anionic peroxidase supposed to be involved in lignification was overproduced and down-regulated in tobacco (Lagrimini, 1991). Plants that overproduced peroxidase synthesized more lignin, but the plants with lower peroxidase activity than the controls did not have less lignin.

CAD has been considered a key enzyme in the lignification pathway because it is the enzyrne that catalyzes the final step in the synthesis of the monolignols, thereby converting the cinnamaldehydes to the corresponding alcohols. Grand et al. (1985) showed that a 90% inhibition of CAD activity in poplar stems by chemical inhibitors re- duces the incorporation of lignin precursors into the polymer by 45%. Brown-midrib mutants in maize (Jorgensen, 1931; Kuc and Nelson, 1964) and sorghum (Bucholtz et al., 1980) show a reduction in lignin content. In the sorghum

bmr6 mutant, the lower lignin content was associated with a reduction in both COMT and CAD activities (Bucholtz et al., 1980; Pillonel et al., 1991). A significant number of coniferaldehyde end groups were detected in the lignin of this mutant by pyrolysis MS (Pillonel et al., 1991) and by thioacidolysis (Chabbert et al., 1993). Also, a more intense phloroglucinol-HC1 staining was detected in this mutant (Pillonel et al., 1991). The maize bml mutant has recently been shown to have a reduction in CAD activity (Holt et al., 1995). Preliminary results also showed a reduced lignin amount and an increased aldehyde content in the lignin of the loblolly pine C A D nu11 mutant (J. MacKay, personal communication).

Reduction of the CAD activity by 93% (Halpin et al., 1994) and by 45% (Hibino et al., 1995) via antisense transformation was described in tobacco and was associated with a red coloration of the xylem. No difference in lignin content was detected using severa1 techniques, but evi- dente for an alteration in lignin composition was demonstrated. A colorimetric detection of aldehydes, based on phloroglucinol-HCI staining, showed a stronger coloration in stem sections of down-regulated CAD plants, suggesting an increased aldehyde content in the lignin. In the study of Halpin et al. (1994) more phenolic compounds could be extracted by NaOH and thioglycolic acid. Also, levels of cinnamaldehydes were increased, especially for syringaldehyde, as analyzed by pyrolysis MS.

We recently cloned a C A D cDNA from poplar (Van Doorsselaere et al., 1995b). Here we report the downregulation of the corresponding CAD in transgenic poplars containing either an antisense or a sense poplar C A D construct. The wood of the poplars with a low CAD activity displayed a red coloration. Despite a strong reduction in CAD activity, the lignin content was not significantly modified in the different transformants. However, the lignin had a higher extractability; more lignin was removed from the wood by alkali extraction and a reduction of the kappa number was obtained after simulated kraft pulping of the down-regulated CAD poplars in comparison with control poplars. This is the first time, to our knowledge, that trees with an improved wood quality that is suitable for industrial applications have been obtained by genetic engineering.

MATERIALS AND METHODS

Transformation of Poplar

The cloning and characterization of a C A D cDNA from poplar (Populus) were recently described (Van Doors- selaere et al., 1995b). The full-length C A D cDNA (cad.Pd X Pt.2) was isolated as a BamHI fragment. This fragment was inserted in both sense and antisense orientation into the pGSJ780A binary vector (harboring a T-DNA region containing a cauliflower mosaic virus 35s promoter-3’T7 and a pNos-NPTII-3’0CS chimeric gene) (Bowler et al., 1991) behind the cauliflower mosaic virus promoter. This resulted in two plasmids, p35SASCAD (the C A D fragment in antisense orientation) and p35SSCAD (the C A D fragment in sense orientation).



Down-Regulation of Cinnamyl Alcohol Dehydrogenase 1481

Agrobacferium-mediated transformation (Leplé et al., 1992) was used to transform poplar (Populus tremula X Populus alba, clone Institut National de Ia Recherche Agronomique no. 717 1-B4). Controls were poplar lines transformed with pBI121 (Jefferson et al., 1987), which is a binary vector containing a cauliflower mosaic virus 35s- GUS and a pNos-NPTII construct.

Plant Material

Three-month-old, greenhouse-grown, regenerated poplars (1.5-2.0 m high; P. tremula x P. alba) were harvested twice a year, in June and in September or October. The stems were cut 15 cm from the base. The lowest 10 cm of the stem was reserved for molecular analysis (enzymatic activity, RNA and DNA analysis) and the other part was used to analyze the lignin characteristics of the wood. The basal part of the stem was left in the greenhouse to allow one shoot to regenerate for the next harvest. For the molecular analysis, bark and phloem were peeled off. The outer xylem (0.5-1 mm) was scraped from the stem with a razor blade and immediately frozen in liquid nitrogen and stored at -80°C.

Preparation of Enzyme Extracts

Scraped xylem was ground in liquid nitrogen. Extraction buffer was added to the powder as described by Goffner et al. (1992), replacing PVPP with 0.4% Polyclar AT (Serva, Heidelberg, Germany). The extract was centrifuged for 15 min at 4°C. The supernatant was used for CAD enzymatic assays. The protein concentration was determined by the method of Bradford (1976) using the dye-binding reagent that was supplied by Bio-Rad.

Assay for CAD Activity

CAD activity was measured, following the oxidation of coniferyl alcohol, based on the method of Wyrambik and Grisebach (1975). Enzymatic assays were performed at 32°C in microtiter plates with 10 to 30 pg of protein. The data presented are the mean CAD activity values in protein extracts derived from three independent grindings. Co- niferyl akohol and NADP were purchased from Sigma and Boehringer Mannheim, respectively.

Native Cel Electrophoresis

CAD isozymes were resolved by nondenaturing PAGE, and CAD enzyme activity was revealed in gel according to the method of MacKay et al. (1995b). The same amount of protein was loaded into each well.

RNA Cel-Blot Analysis

Total RNA was prepared as described by Verwoerd et al. (1989) and quantified spectrophotometrically. RNA (10 pg) was run on 1% agarose gels containing formaldehyde (Ma- niatis et al., 1982) and transferred on a Biodyne B transfer membrane (PALL Europe, Portsmouth, UK) by the alkaline method, as recommended by the manufacturer. The C A D

cDNA was subcloned in pGem2 (Promega) in both orientations, resulting in pGemPOPCADl and pGemPOPCAD2. 32P-labeled, single-stranded riboprobes were synthesized using either SP6 or T7 polymerase (Promega) to make probes that detected either sense or antisense RNA.

A cDNA encoding an isoflavone reductase homolog from poplar was used as a control (W. Boerjan, unpublished results). This cDNA was subcloned in pBluescript SK- (Stratagene) and a 32P-labeled riboprobe was synthesized using T7 polymerase. Prehybridization and hybrid- ization were performed at 65°C in 50% formamide (Mania- tis et al., 1982). Filters were washed in 3X SSC/O.5% SDS ( lx SSC = 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and 0.3X SSC/O.5% SDS at 65°C for 1 h and autoradio- graphed on XAR-5 film (Kodak) with intensifying screens.

Histochemical Stain for Lignin

Carmine-green (Johansen, 1940), phloroglucinol-HC1 (Wiesner reaction; Speer, 1987), and Maule (Iiyama and Pant, 1988) reactions were performed on stem sections of young poplars (50 cm high). Sections of fresh material were made with a hand microtome (Reichert-Jung, Nussloch, Germany). Dark-field pictures were made with a Diaplan microscope (Leitz, Heerbrugg, Switzerland).

Lignin Determination

Lignin content and composition were determined on CWR, which is the dried residue obtained after successive extractions of the freeze-dried and ground wood with tolu- ene:ethanol (2:1, v/v), ethanol, and water. The yields of CWR from the control and transgenic lines were similar and varied between 68 and 77%. Klason lignin content was estimated according to the method of Effland (1977). The content of noncondensed monomeric units was characterized by thioacidolysis, as described by Lapierre et al. (1986).

lsolation and Spectral Characterization of the Dioxane Lignin Fraction

Dioxane lignin fractions were solubilized from CWR according to the method of Monties (1988). Extracts were concentrated prior to precipitation with water; the residue was collected by centrifugation, washed with distilled water, and freeze-dried. This dioxane lignin was resolubilized in dioxane and characterized by UV-visible spectrometry. Alternatively, the dioxane lignin preparation was reduced with NaBH, according to the method of Kirk and Chang (1975), and characterized by UV-visible spectrometry.

Alkali Treatment of the Wood or the CWR

Wood or CWR was subjected to 2 N NaOH at 35°C for 24 h. This resulted in an insoluble SR and a soluble AF. The SR was recovered by filtering through sinter glass filters, followed by extensive washing with water until a neutra1 pH was reached. UV-visible spectra of the AF of wood were recorded from 250 to 500 nm.



1482 Baucher et al. Plant Physiol. Vol. 11 2 , 1996

For the determination of monomeric phenols, the AF was acidified to pH 1.0 to 2.0 prior to ethyl ether extraction of the phenolics from the aqueous phase. Separation of the monomers was achieved by liquid chromatography with a linear gradient of acetonitri1e:water containing 0.1% phosphoric acid. Detection of phenolics was performed at 313 nm (vanillin, syringaldehyde) and 280 nm (p-hydroxybenzoic acid) in a diode array detector; quantification was done using trimethoxycinnamic acid as the intemal standard.

For AL isolation, the AF was acidified to pH 1.0 and allowed to stand at 4°C for 72 h. The precipitate was collected by centrifugation, and the lignin was solubilized in dioxane prior to final precipitation in 1 N acetic acid. AL precipitate was finally recovered by centrifugation, washed with distilled water, and freeze-dried.

Pulp Manufacture

The chemical kraft pulping process is based on alkali extraction and lignin sulfonation. The pulping conditions depend on the percentage of active alkali (Na,S plus NaOH). After pulping, severa1 characteristics of the pulp are investigated: the yield (de Choudens and Valette, 1992); the kappa number (Technical Association of the Pulp and Paper Industry standard, T236cm, 1985), which is an indi- cation of the degree of delignification of the pulp; the DP (International Organization for Standardization standard 5351-1, 1981), which indicates the chain length of the polymer and is an evaluation of the cellulose degradation; and the fiber length (Technical Association of the Pulp and Paper Industry standard, T271pm, 1991). When pulping is performed using a high percentage of alkali, the pulp yield will decrease and both the kappa number and the DP will be lower. Thus, a clear correlation exists between the different characteristics of the pulp and the pulping conditions (Biermann, 1993).

Chemical pulping was simulated in small, pressurized bombs. Anhydrous wood (200-220 g) was used for the experiments and yielded about 100 g of anhydrous pulp after cooking. The chemical impregnation was carried out with 22% active alkali and 25% sulfidity. The temperature was raised to 170°C during 90 min and maintained for 60 min. Then the pulp was washed and screened to extract uncooked particles. Bleaching was performed on 100 g of oven-dried pulp with the O D E/O D sequence, which is a conventional, elementary, chlorine-free bleaching sequence consisting of four delignification steps (de Choudens et al., 1995). The O stage was carried out with 3% NaOH, 0.5% MgSO, during 1 h at 90°C and 10% of pulp consistency under an oxygen pressure of 5 bars. The D stage was carried out at 70°C and 10% pulp consistency with 1.5% C10, for 2 h in the first stage and 0.3% C10, for 4 h in the second stage. The E/O stage was performed with 2% NaOH during 1 h at 70°C and 10% pulp consistency under an oxygen pressure of 2 bars. The pulp was washed with deionized water between each stage of the bleaching sequence and afterward was measured for its brightness. Brightness (International Organization for Standardization standard 2470,1977) describes the whiteness of the pulp on a scale from 0% (absolute black) to 100% (white).

RESULTS

Construction of Sense and Antisense CAD Vectors and Transformation of Poplar

A full-length (1.4 kb) CAD cDNA was inserted either in sense or in antisense orientation into the binary vector pGSJ780A behind the cauliflower mosaic virus 35s promoter, resulting in the plasmids p35SSCAD and p35SASCAD, respectively (Fig. 2). Agrobacterium-mediated transformation of poplar yielded about 20 transformants for both constructs. DNA gel-blot analysis showed that the T-DNA copy number varied between 1 and 5 (data not shown).

Analysis of CAD Activity in Sense and Antisense CAD Poplars

To analyze whether the expression of the chimeric genes resulted in transgenic poplars with a reduced CAD activity, xylem was harvested from young poplars and assayed for CAD activity toward coniferyl alcohol. CAD activity was measured in a11 of the transformants and at different sampling dates. Figure 3a shows the CAD activity of eight antisense transgenic plants that were harvested in June 1994. Three transgenic lines (ASCAD14, ASCAD21, and ASCAD52) had a CAD activity reduced by approximately 70%, compared with the control. The CAD activity of AS- CAD49 was reduced by 40%. Similar reductions were found for these lines at the other sampling dates (data not shown).

As shown in Figure 3b, an enzymatic screening of the sense transformants identified one line (CAD24) with a 30% increase in CAD activity compared with the control value. However, in two transformants (CAD1 and CAD4) CAD activity was down-regulated by approximately 70%. Other parts (bark and leaves) were harvested simulta- neously and assayed for their CAD activities, and in these tissues no significant differences were observed between the antisense and sense transgenic plants and the controls.

ATG

a)

LB PI PI E1 RB

200bp I

p35SSUD - p 3 5 u s c M t-------r

Figure 2. Schematic presentation of sense and antisense CAD constructs. a, Physical map of the poplar CAD cDNA clone. The boxed region represents the coding sequence. The position of the start codon is indicated. 61, BamHI. b, Schematic presentation of the sense and antisense CAD constructs in the vector pGSJ780A. Only the T-DNA region is shown. PI, Pstt; Bt, BamHt; LB, left border; RB, right border; 3’0CS, terminator of the octopine synthase gene; PNOS, nopaline synthase promoter; NPTII, coding sequence of the neomycin phosphotransferase II gene; CaMV, cauliflower mosaic virus; and 3’T7, terminator of the T7 polymerase gene. The arrow at the 61 site indicates the site of insertion of the BI fragments.




pBI121

0,00

u u u u u u u u

Figure 3. CAD enzymatic activity toward coniferyl alcohol of trans-genic and control poplars, a, CAD activity in xylem of differentantisense CAD transgenic and control (pBI121) poplars, b, CADactivity in xylem of different sense CAD transgenic and control(pBI121) poplars. All values are means ± so of three individualmeasurements within the same harvest (June 1994).

The CAD activity in bark and leaves was approximately2-fold lower than in xylem tissue (data not shown).

The results were verified by nondenaturing PAGE fol-lowed by an in-gel CAD activity assay. In xylem extractsfrom control plants, three bands were detected (Fig. 4). Inthe down-regulated CAD lines ASCAD21, ASCAD52, andCADI, the intensity of all three bands was strongly re-duced compared with that of the control.

CAD Transcript Levels in Xylem of Sense and AntisenseCAD Plants

RNA gel-blot analysis was performed on xylem from aselected number of sense and antisense plants using ribo-

Figure 4. Nondenaturing PAGE of xylem proteins from control(pBI121), antisense (ASCAD21 and ASCAD52), and sense (CADI)down-regulated CAD transgenic poplars stained for CAD activity.

probes to detect either the sense or the antisense transcript.As shown in Figure 5a, the endogenous CAD transcriptwas detected in the control and the antisense CAD lines butto a lower extent in the lines ASCAD14, ASCAD21, andASCAD52. The amount of endogenous transcript inASCAD49 was intermediate between the control and thethree lines described above. This result correlated well withthe CAD activity data (Fig. 3a). Antisense RNA could bedetected in only transformant ASCAD49 (data not shown).For the sense transformants, the sense mRNA level (thesum of the endogenous and the transgene CAD mRNAlevels) was similar to the control in CAD9 and in CAD23but was nearly absent in line CADI. A cDNA for an isofla-vone reductase homolog from poplar (K. Vander Mijns-brugge, unpublished results), which is highly expressed inxylem, was used to control the RNA amount that wasloaded onto the gel for each transformant (Fig. 5b).

A smear resulting from RNA degradation was observedin all of our blots with xylem RNA but not with leaf RNA.This might be attributed to RNA degradation during theautolysis of xylem vessels.

Poplars with a Reduced CAD Activity Display a RedColoration of the Xylem

No phenotypic differences in plant growth were ob-served between the control and the transgenic poplars.However, when the bark was peeled off, the xylem of theplants with a reduced CAD activity displayed a red col-oration, the intensity of which was correlated with thedegree of reduction in CAD activity. The most intensecoloration was observed in lines ASCAD14, ASCAD21, andASCAD52 and in CADI and CAD4 transformants; a weakand patchy coloration was observed for lines ASCAD10and ASCAD49. As shown in Figure 6 (A-C), the colorationwas either uniform in the stem of 3-month-old poplars orfollowed patterns (uniform, radial, or concentric). No dis-coloration was detected in the other tissues of the plants.During the second year of growth, the color was seen inonly the newly formed xylem but no longer in the firstgrowth ring (data not shown).

Figure 5. RNA gel-blot analysis of xylem tissue of sense, control, andantisense CAD poplars, a, Total RNA was extracted and hybridizedwith an antisense 32P-labeled riboprobe complementary to the CADcDNA. b, The same filter was hybridized with a 12P-labeled ribo-probe made from a cDNA encoding a poplar isoflavone reductasehomolog. https://plantphysiol.orgDownloaded on November 7, 2020. - Published by

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1484 Baucher et al. Plant Physiol. Vol. 112, 1996

Figure 6. Phenotype of 3-month-old stems of control and CAD down-regulated poplars. A, Stem pieces of differenttransgenic lines and a control showing patterns of red coloration. B, Transverse section of a control poplar. C, Transversesection of a CAD down-regulated poplar (ASCAD21). D, Phloroglucinol-HCI staining of a transverse section of a controlpoplar. E, Phloroglucinol-HCI staining of a transverse section of a down-regulated CAD poplar (ASCAD21). Bars = 1 cm (A),1 mm (B and C), and 100 ;u,m (D and E).

Histochemical Analysis of Stem SectionsAs a first investigation to determine whether reduced

CAD activity in the transgenic poplars was correlated witha modified lignin amount or composition, young stemsections from ASCAD21, ASCAD52, CADI, and controlplants were subjected to different histochemical stainingmethods. No differences were detected using the Ma'ule(specific for syringyl units; Lewis and Yamamoto, 1990) or

the classical carmine-green coloration reactions (data notshown). However, the Wiesner test (phloroglucinol-HCIstaining), which is generally considered to be indicative ofaldehyde end groups (not only the C6-C3 cinnamyl alde-hydes but also the C6-C1 benzaldehydes; Clifford, 1974;Geiger, 1985; Garcia and Latge, 1987; Monties, 1989), re-vealed that the typical pink color of the cell wall ofthe control (Fig. 6D) had become red-brown in the cellhttps://plantphysiol.orgDownloaded on November 7, 2020. - Published by

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wall of CAD down-regulated plants (Fig. 6E). This difference in coloration is indicative of changes in the amount of aldehydes.

Lignin Analysis

Table I shows the lignin content of the CWR of a selec- tion of antisense and sense transgenic poplars and controls that were analyzed in June 1994. The results are mean values of three measurements for one or two plants of the same line. Important variations existed between two repli- cates of a single line. The lignin content of the transgenic poplars was similar to that of the control poplars (Table I).

Structural investigations of the lignin composition were performed by thioacidolysis. With this method the composition of the noncondensed fraction of lignin (i.e. that fraction in which the monomers are linked by the P-O-4-ether linkages) can be determined. The more resistant interunit bonds, i.e. C-C or diaryl ether bonds (condensed fraction), are not broken down. The yields of monomers that were recovered by thioacidolysis (syringyl plus guaiacyl) were not significantly different when lignin from control poplars was compared with lignin from antisense or sense CAD poplars (Table I). Although variation existed, the syringyl! guaiacyl ratio was not significantly altered in the CAD down-regulated poplars (Table I).

Analogous experiments with similar conclusions were performed with antisense lines analyzed at three other sampling dates (data not shown). In general, these data indicate that a reduction of CAD activity by about 70% does not result in a large modification of the lignin content or composition (syringyl! guaiacyl). In addition, no increased amounts of cinnamaldehyde derivatives, which might originate from a possible accumulation of aldehyde precursors, were identified among the thioacidolysis breakdown monomer products (data not shown).

Spectral Evaluation of Dioxane Lignin Fractions from Red and White Poplars

To study the origin of the color observed in the red poplars (CAD down-regulated lines with a red coloration in the xylem), severa1 lignin extraction methods were used (see “Discussion”). Aqueous dioxane is the reagent of choice to solubilize lignin in a neutra1 medium. Dioxane- soluble lignin fractions were isolated from the cell walls of red and white poplars. As shown in Figure 7, a UV-visible spectral comparison of the dioxane lignin of red (AS- CAD52; Fig. 7a) and white (pBI121; Fig. %) poplars showed a higher A,,,-,,, for the red poplar (June 1993). Subsequent reduction of the dioxane lignin by NaBH,, followed by spectral comparison, resulted in a comparable spectrum for both red and white poplars. Such UV-spectral behavior indicates an increased amount of conjugated aldehyde moieties in the lignin from red poplars (Higuchi et al., 1994). Similar spectra were obtained with the other red poplars (data not shown).

Cell Wall Reactivity to Alkali

Mild alkali treatment of the CWR results in the ionization of free phenolic functions and allows the cleavage of weak linkages between lignin and polysaccharides associated in lignin-polysaccharide complexes. A mild alkali treatment was applied to CWR from selected white and red poplars harvested in September 1993, allowing the recov- ery of an insoluble SR and a corresponding solubilized AF (see “Materials and Methods”). Table I1 shows that the SR of the red poplar wood contained a lower amount of lignin (as measured by the Klason method) than the corresponding SR obtained from white poplars. Yields of SR were of the same order from both types of poplar. Spectral characterization of the AF indicated a higher relative AZs0 and

Table 1. Lignin content and composition of xylem CWR from transgenic CAD and control poplars from )une 7994 harvest

When available, measurements were performed on two individual plants for the same line (e.g ASCAD3-1 and ASCAD3-2). Controls were pB1121 transgenic poplars. Data are means 5 SD of three experiments and are expressed as weight percentages for Klason lignin ( K L ) expressed as percentage of CWR and as pmol g-’ CWR for G (guaiacyl unit) and S (syringyl unit) yields.

Line K L c 5 5/c S+C

Antisense CAD poplars pB1121 ASCAD3-1 ASCAD3-2 ASCAD8 ASCADl O ASCAD14 ASCAD2l-1 ASCADZl -2 ASCAD49 ASCAD52-1 ASCAD52-2

Sense CAD poplars pBI121 CAD1 CAD9 CAD23

18.4 2 0.1 19.5 t 0.2 18.5 2 0.1 20.6 2 0.1 18.3 2 0.1 19.5 t 0.1 18.6 2 0.2 18.4 t 0.1 17.7 t 0.2 20.3 2 0.2 18.9 2 0.1

18.4 2 0.1 18.7 2 0.1

18.8 2 0.2 -

153 2 6 133 2 3 145 2 2 151 2 1 140 2 3 136 2 4 131 2 2 135 2 2 154 I 2 126 2 4 1 1 7 2 1

153 2 6 116 I 2 154- t 1 145 2 3

261 2 16 200 2 2 233 2 1 239 -t 1 273 2 6 233 2 7 173 I 3 183 2 2 253 2 8 196 2 6 207 2 4

261 I 16 211 t 7 271 2 2 256 2 7

1.71 1.50 1.61 1.58 1.95 1.71 1.32 1.36 1.64 1.56 1.77

1.71 1.82 1.76 1.77

414 t 22 333 2 5 378 t 3 390 2 2 413 t 9 369 2 11 304 t 5 318 t 4 407 2 10 322 2 10 324 2 5

414 2 22 327 t 9 425 2 3 401 +- 10

a -, Not determined.



1486

A 1.0 1- Baucher et al. Plant Physiol. Vol. 112, 1996

Figure 7. UV-visible absorption spectra of dioxane lignin from red (ASCAD52; a) and white (pBI121; b) transgenic poplars before (- ) and after (- - - -) NaBH, reduction (June 1993).

1 'L I A,,,-,,, for the AF from red poplar (ASCAD52) compared with that of white poplar (pBI121) (Fig. 8, September 1993). A higher AZa0 is indicative of the presence of larger amounts of phenolics and the is indicative of the occurrence of alkali-labile conjugated phenolics (Adler and Marton, 1959). As shown in Table 111, subsequent HPLC fractionation of the AF revealed higher levels of vanillin and mainly syringaldehyde (C6-C1 aldehydes) in the extracts from red poplars than in those from control and white antisense C A D poplars. No significant differences were found in the leve1 of p-hydroxybenzoic acid that were released by the alkali treatment. Transgenic plants with only 40% reduction in CAD activity (ASCAD49) did not show any increase in C6-C1 aldehydes. No significant quantities of the corresponding C6-C3 cinnamaldehydes were detected. Comparable results were obtained in the SR and in the AF from plants harvested in October 1994 (data not shown).

To further investigate the composition of the AF from red and white poplars, the AF fraction was acidified to precipitate and further isolate an acid-insoluble lignin (AL) fraction. Whereas it was nearly impossible to recover some AL precipitate from the white poplar AF, substantial amounts of AL were precipitated from the AF from the red poplars (Table IV). Similar results were obtained in Octo- ber 1994. From these results we conclude that the wood of

Table II. Lignin content of CWR and yield and lignin content of SR from the CWR of white and red poplars from September 1993 harvest

means t SD of three experiments. Klason lignin (KL) is expressed as percentage of CWR. Data are

Line KL of CWR Yield SR KL of SR

% % CWR % White poplars

pBll21 18.3 t 0.1 60.4 t 0.2 24.5 t 0.1 ASCAD8 19.2 t 0.5 63.4 t 0.3 23.2 t 0.2 ASCAD9 17.6 t 0.1 61.7 t 0.1 23.9 t 0.1

ASCAD21 18.2 t 0.4 56.0 t 0.7 18.3 t 0.1 ASCAD52 17.6 t 0.1 60.9 t 0.9 20.9 t 0.2

Red poplars

300 400 500

A (nm)

Figure 8. Absorption spectra of an NaOH extract from white (pBI121; 1 ) and red (ASCAD52; 2) poplar xylem (September 1993). A, Wavelength.

red poplars contains a higher quantity of lignin that is soluble in alkali and can be precipitated by acidification.

Pulp Production and Pulp Bleaching of Transgenic CAD Poplars

Chemical pulping trials were carried out on the 3-month- old transgenic poplars. No significant difference in pulp yield or in fiber length was observed between red and white pulps (Table V). However, a reduction in kappa number (up to 22%) was observed for lines ASCAD14, ASCAD21, ASCAD49, and ASCAD52 with similar DP values, indicating that more lignin is extracted from the red poplars but that the polysaccharides are degraded to a similar degree as in the control poplars.

During paper making, the pulping process is followed by a bleaching process to remove the residual lignin from the pulp, which results in a reduction of the pulp yield but an increase in brightness. Equally, the cellulose DP value de-

Table 111. Yields of phenolic compounds released from red and white poplar xylem by NaOH extraction from September 1993 harvest

three experiments. Results are expressed as pmol g-' CWR. Data are means t SD of

p-H ydroxybenzoic Acid

Line Vanillin Syringaldehyde

White poplars 1.2 t 0.2 13.6 t 0.5

ASCAD3 1.8 f 0.1 1.2 t 0.1 23.2 t 0.1 23.9 t 0.5 ASCAD8 1.7 t 0.1 1.3 t 0.1

ASCAD9 1.6 f 0.1 1.2 2 0.1 16.7 2 0.2

ASCAD14 2.2 t 0.1 5.4 t 0.1 21 .o t 0.1 21.5 2 0.9 ASCAD21 4.3 t 0.1 14.7 ? 0.1

ASCAD49 1.6 t 0.1 1.5 t 0.1 18.2 ? 0.8 ASCAD52 2.2 t 0.1 7.6 2 0.1 20.8 t 1.4

pBll21 1.9 2 0.1

Red poplars




Table IV. Yield of AL issued from control and red poplar xylem CWR from September 7 993 harvest

Values of Klason lignin ( K L ) are given in Table II. Line Yield of AL

% K L pB1121 <l ASCAD2l 11.0 2 0.3

. ASCAD52 5.0 2 0.4

creases because the bleaching agents attack the cellulose polymer. The bleachability for both pulps was comparable (data not shown).

DISCUSSION

We have reduced the CAD activity in xylem tissue of transgenic poplar trees by expressing a poplar C A D cDNA in sense and antisense orientations. This enzymatic inhibition was correlated with a red coloration of the xylem. Although the lignin content of the transgenic lines was similar to that of the control lines, its structure was modified, as shown by its higher extractability in alkali and in kraft pulping experiments.

In several sense and antisense C A D transgenic poplar lines, the CAD activity in xylem was reduced by approximately 70%. By native gel electrophoresis the intensity of the three bands detected in the control was reduced in the down-regulated poplars. CAD has been shown to be detected electrophoretically in multiple forms in Salix spp. and in other plants (Mansell et al., 1976). Because the poplar clone that we have used is a hybrid and poplar CAD is active as a dimer (Sarni et al., 1984), the upper and lower bands might correspond to the homodimers and the mid- dle band might correspond to the heterodimer. A similar situation exists in pine (MacKay et al., 199513).

Despite the large reduction of the CAD activity in the xylem, wild-type CAD activity levels were detected in bark and in leaves. This could be due to the presence of another CAD activity in these tissues. Two isoforms, CADl and CAD2, that differ in their amino acid sequences, their substrate specificities, and their molecular weights have been purified from Eucalyptus xylem (Goffner et al., 1992) and periderm (Hawkins and Boudet, 1994). Alternatively, the efficiency of the antisense effect may vary, depending

on the endogenous transcript level in a particular tissue or the developmental stage of the plants (Atanassova et al., 1995; De Lange et al., 1995). Indeed, the endogenous C A D transcript level is higher in xylem than in bark and leaves (M. Baucher, unpublished results).

Red Xylem

A red coloration of the xylem seems to be typical of plants with a reduced CAD activity. In the antisense C A D transgenic tobacco plants analyzed by Halpin et al. (1994) and by Hibino et al. (1995), this phenotype was correlated with a residual activity of 7% but not of 20%, and of 55% but not of 80%, respectively. In antisense C A D poplar (this study) and in antisense C A D transgenic alfalfa (M. Baucher, unpublished results), the red coloration was detected below 60% residual CAD activity. The maize bml (Jorgensen, 1931; Kuc and Nelson, 1964; Holt et al., 1995), the sorghum bmu6 (Bucholtz et al., 1980), and the pine (MacKay et al., 1995a) mutants with low CAD activity also show a red coloration of the xylem.

It is interesting that the maize bm3 mutants (Jorgensen, 1931; Kuc and Nelson, 1964), which have structural modi- fications of the COMT gene (Vignols et al., 1995), and the antisense COMT transgenic poplars (Van Doorsselaere et al., 1995a) show a similar coloration in the vascular tissue. These data indicate that a reddish coloration can be caused by defects in different genes (COMT and/or CAD).

The red coloration is unstable. In the study of Halpin et al. (1994) the red color of the stem was visible during the growth of the plants but disappeared at the flowering stage. In the bm mutants the color may disappear when maturation is achieved (Jorgensen, 1931). In transgenic poplars the red color was intense during the first growing season but disappeared or was much paler during the winter. In the second year of growth the red color was restricted to the young xylem and disappeared in the first growth ring. The reason for the disappearance of the color is not known. There are several indications that the red color of the CAD down-regulated plants might be due to the presence of conjugated aldehydes in the lignin. By an unknown mechanism, the degree of conjugation may be reduced during aging, resulting in the disappearance of the red color (see below).

Table V. Pulping characteristics of 3-month-old white and red poplar trees from September 1993 harvest

Line KLa kappa no. DP Yield Fiber Length

% % mm

pB1121 19.2 20.8 1010 47.9 0.42 ASCAD3 19.5 21.1 1 O90 39.4 0.40

ASCADl4 18.9 16.5 1095 44.7 0.40 ASCAD2 1 18.4 19.2 1140 47.5 0.43 ASCAD49 19.0 17.7 1127 45.3 0.38

White poplars

Red poplars

18.9 18.8 990 43.4 ASCA D 5 2 - b

a KL, Klason lignin. -, Not determined.



i 488 Baucher et al. Plant Physiol. Vol. 1 1 2, 1996

Lignin Amount

In contrast to the results obtained in poplar stems treated with CAD inhibitors and in CAD bml and bm6 mutants, the transgenic poplar and the transgenic tobacco had a similar lignin content compared with that of controls. These results suggest that CAD may become a rate-limiting step for the amount of lignin if the inhibition in its activity is strong enough. This activity leve1 might be different in different species.

Lignin Composition

The monomeric composition of lignin, as determined by thioacidolysis, showed no significant modification of the syringyl / guaiacyl ratio in down-regulated CAD poplars. This is in contradiction to the results obtained in down- regulated CAD tobacco plants by Halpin et al. (1994) and Higuchi et al. (1994), who showed a reduction and an increase in the syringyl/ guaiacyl ratio, respectively. The bml CAD mutant had a higher syringyl/guaiacyl ratio than the wild type (Barrière et al., 1994). In our analysis consid- erable variation was observed between the different transgenic poplar lines and controls, but we were unable to observe significant differences in lignin composition. A detailed analysis throughout the growing season confirmed that the down-regulated CAD plants (ASCAD21 and ASCAD52) and the control had comparable lignin compositions (syringyl/ guaiacyl and syringyl plus guaiacyl; L. Jouanin, unpublished results).

Severa1 attempts were undertaken to isolate an enriched red-colored fraction from the wood. Extracts that were recovered from the red wood by acetyl bromide or trieth- ylene glycol (lignin characterization), sulfuric acid (determination of phenols and quinones linked to hemicellulose), butanol/ HC1 (proanthocyan determination), or methanol (anthocyan determination) showed no significant differences between A,,, and A,,, as compared with extracts obtained from white wood, suggesting that the red chro- mophoric groups were probably unstable and not solubilized (B. Chabbert, unpublished results).

Evidence for lncorporation of Aldehydes in the Lignin

The presence of aldehydes in the lignin polymer from red wood was suggested by several experiments. First, although not completely conclusive, phloroglucinol-HC1 staining was markedly different on stems from red poplars than from white poplars.

Second, spectral analysis (250-500 nm) of the dioxane lignin from the red poplars showed a higher A,,,~,,, than white poplars. Reduction of both of these lignin fractions by NaBH, resulted in a comparable spectrum. These data indicate the presence of larger amounts of conjugated aldehyde moieties in the lignin from red poplars (Adler and Marton, 1959).

Third, dehydrogenative polymerization of coniferyl alcohol results in a pale-brown-colored precipitate, whereas dehydrogenation polymers from coniferyl alcohol plus coniferaldehyde or from coniferaldehyde alone result in a red polymer (Higuchi et al., 1994; Tollier et al., 1995). The

incorporation of aldehydes in the lignin polymer would generate a highly conjugated polymer that might explain the red coloration of the xylem (Higuchi et al., 1994).

Thioacidolysis from dehydrogenation polymers made from coniferaldehyde allows aldehyde products to be detected (Higuchi et al., 1994; Tollier et al., 1995). No cinnamaldehydes were detected from the red poplar (this study), from the antisense CAD transgenic tobacco (Halpin et al., 1994), or from the bml mutant (Chabbert et al., 1993) via thioacidolysis. These results are in contradiction to the results obtained by Higuchi et al. (1994), who detected increased amounts of aldehydes among the thioacidolysis products from down-regulated CAD tobacco plants. It is possible that during the development of the plant the aldehydes can be detected only at a certain developmental stage. Evidence for changes in the lignin characteristics over time was observed in this study by the disappearance of the red color during development of the tree.

Higher Extractability of the Lignin

It is interesting that the lignin of the CAD down- regulated red poplars had an altered reactivity toward alkali when compared with that of white poplars: less Klason lignin remained in the SR and a significant amount of lignin could be precipitated in the AF. In addition, increased levels of benzaldehydes, mainly syringaldehyde, were detected in the AF.

The higher extractability of lignin upon alkali treatment might occur for several reasons. The incorporation of aldehydes into the lignin polymer would (a) render the free hydroxyl group at the C4 position more susceptible to ionization in alkali, and (b) render the a and the p carbons more susceptible to chemical attack by alkali. This would not only explain the higher solubility of the lignin but also the higher amounts of the C6-C1 benzaldehydes, vanillin, and syringaldehyde. Only small quantities of these aldehydes are present in wild-type lignin (Grisebach, 1981; Fengel and Wegener, 1984; Higuchi, 1985; Monties, 1989; Lewis and Yamamoto, 1990). The increased levels of these C6-C1 compounds in the down-regulated CAD poplars are thus probably derived from C6-C3 aldehydes. An addi- tional reason why the lignin would be more extractable is that the y-terminal alcohol groups might be involved in the formation of cross-links with polysaccharides (Monties and Lapierre, 1981; Ralph et al., 1994). Replacing part of these alcohol groups with aldehyde groups may result in a reduced frequency of cross-links to polysaccharides.

Pulping

The kraft pulping data indicated a reduction of the kappa number without significant difference in cellulose degradation in the pulps from red poplars as compared with those from white poplars. Because the Klason lignin content was similar for both types of plants, such data are indicative of qualitative, rather than quantitative, modifi- cations of the lignin from the transgenic poplars. These results, obtained from 3-month-old poplars, hold great promise for the pulp and paper industry. As outlined by




Sederoff e t al. (1994), even small improvements i n the processing of wood for pulp a n d paper can be of high value because of the large scale of the industries.

Kraft pulping experiments on 2-year-old CAD down- regulated poplars confirmed the decrease i n kappa number (M. Petit-Conil, unpublished results). However, it still needs to be demonstrated that this effect is maintained for a t least 7 to 10 years, the age at which poplars are harvested for pu lp and paper production. Field trials have been es- tablished to evaluate the pulping and bleaching characteristics during the development of the tree.

ACKNOWLEDCMENTS

The authors are indebted to Frank Jajik and Marie-Claude Sou- rie for technical assistance, Catherine Lapierre for critica1 views on thioacidolysis, Patrick Zimmer and Gérard Vastra for the mainte- nance of the plants in the greenhouse, Eric Van der Eycken for fruitful discussions, and Martine De Cock, Karel Spruyt, and Christiane Germonprez for help with the manuscript.

Received May 9, 1996; accepted August 30, 1996. Copyright Clearance Center: 0032-0889/96/ 112/1479/ 12.

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